Abstract 6387: Inhibition of Lymphoid Enhancer Factor1<i>(LEF1</i>) in acute myeloid leukemia overcomes acquired cytarabine resistance
Authors: Saswati Das, Raveen Stephen Illangeswaran, Daniel Zechariah Paul Jebanesan, Rakhi Thalayatta Vidhyadharan, Nayanthara Karpillymoola Bijukumar, Bharathi Murugan Rajamani, Martin Michaelis, Jindrich Cinatl, Vikram Mathews, Shaji Ramchandran Velayudhan, Poonkuzhali Balasubramanian
Journal: Cancer Research (2022)

Abstract Canonical Wnt-β-catenin signalling is frequently dysregulated in myeloid leukemia. LEF1 (Lymphoid Enhancer Factor 1), a key mediator of Wnt signalling, functions as an interacting partner with β-catenin. Previous studies have shown that LEF1 is associated with leukemic transformation and is an independent prognostic factor in normal karyotype AML. Through high throughput transcriptome profiling between the parental and acquired cytarabine resistant cell lines, we identified that LEF1 to be a potential target mediating this resistance. Here we evaluated the role, of molecular and pharmacological inhibition of LEF1 in overcoming acquired cytarabine resistance. The differential expression of LEF1 in THP1, MV4-11 and U937 parental and Ara-C resistant AML cell lines was checked by quantitative RT-PCR, immunoblot and immunofluorescence. LEF1 knock-out (KO) cells were generated using the CRISPR-Cas9 in the THP1 cytarabine resistant cell line. Lentiviral based over-expression of LEF1 was carried out in THP1 parental cell line to ensure that LEF1 promotes Ara-C resistance. For nuclear translocation of LEF1, the small molecule CHIR99021 was used in over-expressed cells.The knock-out and over-expressed cells were characterized by analysing proliferation, cell cycle and cytotoxicity assay. The effect of pharmacological inhibition of LEF1 using niclosamide was examined by in-vitro-cytotoxicity assay.Although LEF1 transcript expression was high in all the three cytarabine resistant cell lines compared to the parental cell lines, THP1-araC-R cell line showed around ≈ 500-fold change in expression and hence was used for further experiments. THP1-araC-R was marginally cross resistant to both DNR (IC50 Parent -0.19µM, AraC 0.26µM) and ATO (IC50 Parent -2.1µM, AraC &amp;gt;4µM). LEF1 knock-out showed complete absence of LEF1 protein, decreased rate of proliferation and improved sensitivity to Ara-C (Ara-C IC50- 1430 µM in the untransduced vs. 387.4 µM after LEF1 KO). Over-expression of LEF1 with a fold change of ≈1000 followed by enforced nuclear translocation using the small molecule CHIR99021 in parental cell line resulted in increased resistance to Ara-C (IC50 Parental- 50.7µM vs OE CHIR treated 240.05µM). Further, niclosamide, the pharmacological inhibitor of LEF1 was able to re-sensitize the THP1 AraC -resistant cells (IC50: 1430 µM for UT vs 516.8 µM treated with niclosamide). Our results suggest that inhibition of LEF1 by molecular and pharmacological means showed considerable increase in cytarabine sensitivity in-vitro while we are testing its effect in-vivo using a transplantable AML mouse model. Citation Format: Saswati Das, Raveen Stephen Illangeswaran, Daniel Zechariah Paul Jebanesan, Rakhi Thalayatta Vidhyadharan, Nayanthara Karpillymoola Bijukumar, Bharathi Murugan Rajamani, Martin Michaelis, Jindrich Cinatl Jr., Vikram Mathews, Shaji Ramchandran Velayudhan, Poonkuzhali Balasubramanian. Inhibition of Lymphoid Enhancer Factor1(LEF1) in acute myeloid leukemia overcomes acquired cytarabine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6387.